In recent years a growing number of foreign governments and radical groups, many unfriendly to the United States and its allies, have added Bacillus anthracis, the cause of anthrax, to their arsenals of weapons of mass destruction. This bacterium is convenient for use in warfare and terrorism because it is highly pathogenic, can be grown easily, and forms spores. The spore's resistance to extreme temperatures, noxious chemicals, desiccation, and physical damage make it suitable for incorporation into explosive weapons and for concealment in terrorist devices. Spores can enter the body through ingestion or by inhalation, germinate into vegetative cells, and cause death within 1 to 7 days, sometimes with little or no overt sign of infection preceding death. Antibiotics can be used to treat anthrax, but B. anthracis strains have been, or can be, constructed that resist these drugs. A vaccine against an anthrax toxin exists, but it is slow acting and difficult to produce. Thus, better responses to the threat of anthrax are needed. The overall goal of this proposal is to identify protein factors on the surface of the B. anthracis spore that contribute to pathogenesis and are potential targets for new vaccines and drug intervention. The outermost layer of the B. anthracis spore is a semi-permeable, loose-fitting, balloon-like structure called the exosporium. Approximately 50 percent of the exosporium is composed of a protein consisting of perhaps eight unique protein components. At present, no integral exosporium proteins of B. anthracis have been identified. However, growing evidence suggests that they will contribute significantly to key spore properties such as structure, viability, germination, infectivity, and virulence. In the proposed research, we will identify the proteins in the B. anthracis exosporium and attempt to assign a function to each. The three specific aims of this proposal are: (1) Identify the proteins present in the exosporium of B. anthracis and the genes that encode these proteins. This will be accomplished by isolating proteins, determining partial sequence by mass spectrometry, and identifying proteins and their genes with the aid of the sequenced B. anthracis genome; (2) Mutationally inactivate the genes encoding the exosporium proteins and examine the effects on the key spore properties listed above; and (3) Use an existing panel of monoclonal antibodies against the B. anthracis spore surface (i.e., exosporium) to create affinity-matured single chain antibodies that neutralize exosporium protein activity, then test the effects of these reagents on spore properties.